Human Serum Processing and Analysis Methods for Rapid and Reproducible N-Glycan Mass Profiling Scott R. Kronewitter,† Maria Lorna A. de Leoz,† Kyle S. Peacock,† Kelly R. McBride,† Hyun Joo An,† Suzanne Miyamoto,‡ Gary S. Leiserowitz,‡ and Carlito B. Lebrilla*,† Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616, and Division of Hematology/Oncology, UC Davis Cancer Center, 4501 X Street, Sacramento, California 95817 Received March 5, 2010
Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. Understanding the variability of the measurement devices helps understand the variability associated with the chemical processing. This report explores the potential use of methods expediting the enzymatic release of glycans such as a microwave reactor and automation of the solid-phase extraction with a robotic liquid handler. The study employs matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry but would be suitable with any mass spectrometry method. Methods for system-wide data analysis are examined because proper metrics for evaluating the performance of glycan sample preparation procedures are not well established. Keywords: glycomics • interpretation of mass spectra • Fourier transform ion cyclotron resonance mass spectrometry • human serum
Introduction Glycans are complex linear and branched arrangements of monosaccharides that are essential to the vitality of organisms. It is projected that over 50% of all proteins are glycosylated based on the Swiss-Prot database of known protein sequences and putative binding sites.1 N-linked glycans make up the most abundant group and are found only on an asparagine that is adjoined by a variable amino acid followed by serine, threonine, or less commonly cysteine amino acid (Asn-Xaa-Ser/Thr/Cys).2–4 N-linked glycans have a large diversity in structures bound to a consensus sequence on a glycoprotein. The biological importance of glycosylation in cellular recognition and communication has made them a prime target for discovering biomarkers for diseases.5–10 N-glycans are typically isolated for analysis by cleaving off the glycan from glycoproteins with the endoglycosidase enzyme PNGase F (peptide: N-Glycosidase F). PNGase F is often the enzyme of choice because of its broad specificity toward almost all N-glycans. However, if the N-glycan core is modified with an R1-3 fucose, it will be resistant to PNGase F.11 After release, mass spectrometry is often performed to obtain glycan compositions. Mass spectrometry is currently the best method for * To whom correspondence should be addressed. Carlito B. Lebrilla, e-mail:
[email protected]. Telephone: 1-530-752-0504. Fax: 1-530-7528995. Postal Address: Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California 95616. † University of California Davis. ‡ UC Davis Cancer Center.
4952 Journal of Proteome Research 2010, 9, 4952–4959 Published on Web 08/10/2010
profiling glycans due to its sensitivity and speed of analysis. Furthermore, profiling glycan compositions is one key step toward understanding structure-function relationships of specific glycan structures. Biomarker discovery relies on the reproducible release and quantitative analysis of glycans from tissue. The most direct method for discovery involves three major steps: (1) the release of the glycan, (2) enrichment, and (3) mass spectrometry. The reliability of the markers relies on each of these steps to be highly reproducible. Global enzymatic release of N-glycans as performed with PNGase F requires treatment of serum in a physiological buffer and reacted isothermally at 37 °C. The shortest amount of time for digestion of pure glycoproteins is 2 h,12,13 whereas digestion of complex mixtures, like serum, can take from 12 to 22 h.14–16 However, three approaches have been implemented to alter the common PNGase F enzymatic release method: the creation of an immobilized PNGase F enzyme reactor,17 high pressurecycling of reaction conditions,18 and the implementation of a microwave reactor.15,19 Palm et al.17 built a monolithic PNGase F enzyme micro reactor by immobilizing the enzyme covalently onto a porous polymer-based monolith. They immobilized the PNGase F enzyme in a cross-linked acrylamide gel. Standard glycoproteins (chicken ovalbumin, bovine asialofetuin, bovine ribonuclease B, and bovine R1-acid glycoprotein) were shown to effectively deglycosylate in 3.5 min incubation times while maintaining constant activity for eight weeks. 10.1021/pr100202a
2010 American Chemical Society
Rapid and Reproducible N-Glycan Mass Profiling a
Table 1. Sample Breakdown
Sample Sample Sample Sample
Set Set Set Set
A B C D
samples
source
release method
4 4 1 48
Sigma Sigma Sigma GOG
Water Bath Microwave Microwave Microwave
solid phase extraction
Vacuum Manifold Gilson GX-274 Gilson GX-274 Gilson GX-274
a
Set A,B,C came from a single human serum sample purchased from Sigma Aldrich. Set D consists of 48 different healthy control individuals purchased from the Gynecologic Oncology Group.
Szabo et al.18 used pressure cycling technology to enhance the release of N-glycans by PNGase F digestion. Pressure cycling between low (atmospheric) and high pressures (up to 30 kpsi) was shown to enhance the glycosidase activity requiring minutes for glycan release while requiring relatively low amounts of enzyme. Sandoval et al.19 employed microwave irradiation to enhance the enzymatic activity. Glycoproteins Herceptin (trastuzumab), Avastin (bevacizumab) (Genentech Inc.) and bovine ribonuclease B were deglycosylated with reaction times of 10 min. Bereman et al.15 experimented with a liquid cooled microwave reactor that allowed for temperature control. They found that a fixed temperature of 25 °C for 20 min (∼250 W) had the best performance based on the sum of the plasma glycan abundances. This report examines a high-throughput sample preparation pipeline for global glycan mass profiling. It includes several metrics for method analysis including the percent coverage of glycan libraries, average glycan abundances, and coefficients of variation of glycan abundances. By isolating technical variables and experimental variables from sample variability, the variation of the glycan processing steps and spectra collection were studied. Microwave assisted enzymatic Nglycan release and automated solid phase extraction (performed by a liquid handler) were implemented in this workflow to speed up and stabilize the processing. A matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometer was used to rapidly scan the glycan sample and capture high mass accuracy highresolution mass spectra. The expedited automated sample preparation and mass spectra collection methods scale well for large clinical sample sets allowing for fast turnaround.
Experimental Section Samples. The human serum samples were purchased from Sigma Aldrich (Sigma) and the Gynecological Oncology Group (GOG) tissue bank. The number of technical replicates in the case of Sigma serum, and number of individual serum samples from the GOG are shown in Table 1. The pertinent sample processing parameters are also included. Human serum samples used for reproducibility studies were purchased from Sigma and all came from the same person and lot (Sets A,B,C) while samples for the broad human profile study (Set D) came from the GOG tissue bank. The GOG samples were organized and divided into aliquots at the University of California Davis Medical Center Clinical Laboratories. The serum processing at the GOG tissue bank was performed with an Internal Review Board (IRB) approved protocol. All samples were transported frozen and stored in a -75 °C freezer prior to processing. Evaluation of the reproducibility of MALDI sample spot preparation within a spot (intraspot), and between spots (interspot) was performed on a single sample (Set A). Using a
research articles single sample decoupled the sample preparation variability from the variability due to mass spectra collection. Process reproducibility is evaluated with coefficients of variation (CV) and sample correlation coefficients. Standardization of the mass spectra collection process was also optimized on a set of four technical replicate serum samples. A human serum sample set of four technical replicates from one serum sample were used to compare two sample processing methods and isolate the biological diversity from the experiments. The standard method (Set B) involves typical isothermal PNGase F N-glycan enzymatic release and manual vacuum manifold solid-phase extraction. The new method (Set C) replaces the glycan release with a more streamlined microwave-assisted PNGase F N-glycan release and exchanges the vacuum manifold with an automated Gilson GX-274 ASPEC liquid handler. This method was further applied to a large set of healthy human serum samples (n ) 48) and evaluated (Set D). Glycan profiles were employed for comparing spectra across large sample sets. Metrics used here for method evaluation include how often a glycan can be detected in a sample set and its average abundances. Protein Denaturation. Serum proteins were denatured under mild conditions to cleave disulfide bonds while minimizing glycan degradation. Serum samples were mixed in equal parts with an aqueous solution of 200 mM ammonium bicarbonate and 10 mM dithiothreitol. Detergents and buffers used during this step can often interfere with the mass spectrometry analysis downstream.20 The ammonium bicarbonate/dithiothreitol solution was chosen because it is easily removed during the solid-phase extraction step. Low salt levels produce samples that are easily ionized in the mass spectrometer. The thermal denaturation includes four cycles of alternating between boiling water (100 °C) and room temperature (25 °C) water for 15 s each. The whole process takes 2 min per set of 24 samples, which is favorable for high throughput applications. Rapid Enzymatic Glycan Release. The CEM microwave reactor (CEM Corporation, North Carolina) has two operational modes: standard and solid-phase synthesis (SPS) mode. Both modes require the user to specify a temperature and microwave power set point. The temperature is monitored by a fiber optic probe and both temperature and microwave power levels are presented on the reactor display. Heat generated from the microwave radiation was removed by a stream of air around a water-filled sample cup. The water in the cup surrounds the sample tubes allowing for a mediated constant temperature across all samples. For the standard mode temperature control, the reactor uses process control algorithms to vary the microwave power to hold a selected temperature. Unfortunately, the algorithms are designed for high temperature reactions (i.e., organic reactions). Low temperature conditions produce a sinusoidal response in microwave power and sample temperature. The variability in power supplied to the sample from this method makes studying the microwave enhancement effect difficult. Standard mode temperature controlled microwave power and sample temperature vs. time were plotted and presented in Figure 1a. Alternately, the SPS mode has a temperature set point and uses a step function of power inputs to maintain the temperature. By relaxing the temperature set point in the SPS mode, the step function can be avoided so that well-controlled, constant power experiments can be conducted. Each constant power setting has a different equilibrium temperature that is balanced by the cooling capacity Journal of Proteome Research • Vol. 9, No. 10, 2010 4953
research articles
Figure 1. (a) Sinusoidal response of microwave power (watt) and sample temperature (degree Celsius) under standard mode reactor operation and a 37 °C set point. (b) PNGase F enzymatic digestion temperature profiles. The solid line represents the standard water bath incubation at 37 °C. The 6W and 20W aircooled microwave reactor temperature profiles are presented. The temperature curves reach their equilibrium temperature eight minutes into the run.
of the reactor. Figure 1b depicts two stable equilibrium temperatures at 30 °C (6W) and 44 °C (20W). The optimal water bath temperatures for PNGase F release are plotted as a horizontal line. For a 10 min run, a 6W setting will input 3.5 KJ/run of microwave energy, while the 20W setting will input 12 KJ/run. However, much of this energy will be absorbed into the reactor walls and cooling water so that a 3-fold energy increase will keep the temperatures at a reasonable range (
